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The Methodology Used to Measure Differential Gene Expression Affects the Outcome

¹ Division of Hematology/Oncology, Department of Medicine, and
² Departments of Preventive Medicine,
⁴ Urology, and
⁵ Pathology, Northwestern University Medical School and the Robert H. Lurie Cancer Center of Northwestern University, Chicago, IL;
³ Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE

Correspondence: Raymond C. Bergan, Division of Hematology/Oncology, Northwestern University, Olson 8321, 240 East Huron St., Chicago, IL 60611–3008 (phone: 312–908–5284; fax: 312–503–4744; email: r-bergan{at}northwestern.edu).

Confirmation of gene expression by a second methodology is critical in order to detect false-positive findings associated with microarrays. However, the impact of methodology upon the measurement of gene expression has not been rigorously evaluated. In the current study, we compared differential gene expression between PC3 and PC3-M human prostate cancer cell lines using three separate methods: microarray, quantitative RT/PCR (qRT/PCR), and Northern blotting. The PC3 to PC3-M ratio of gene expression was determined for each of 24 different genes evaluated, by each of the three methods. Comparison of gene expression ratios between Northern and microarray, Northern and qRT/PCR, and microarray and qRT/PCR, gave correlation coefficients (r) of 0.72, 0.39, and 0.63, respectively. In each instance, one to two outlier genes were apparent. Their exclusion from analysis gave r values of 0.79, 0.72, and 0.83, respectively. These findings demonstrate that the assessment of differential gene expression is dependent upon the methodology used in each situation where outcome between different methodologies was compared, the presence of a relatively limited number of outlier genes precludes high overall correlation between the methods. Validation of gene expression by different methods should be performed whenever possible.

Key Words: microarray • quantitative PCR • Northern blot • gene expression